Biology subjects

Collins, K.

Publications and source records attributed to Collins, K..

30 records · Page 2Linked to original sources

bioRxiv2023

Streamlined and sensitive mono- and diribosome profiling in yeast and human cells

Ribosome profiling has unveiled diverse regulations and perturbations of translation through a transcriptome-wide survey of ribosome occupancy, read out by sequencing of ribosome-protected mRNA fragments. Generation of ribosome footprints and their conversion into sequencing libraries is technically demanding and sensitive to biases that distort the representation of physiological ribosome occupancy. We address these challenges by producing ribosome footprints with P1 nuclease rather than RNase I and replacing RNA ligation with Ordered Two-Template Relay, a single-tube protocol for sequencing library preparation that incorporates adapters by reverse transcription. Our streamlined approach reduced sequence bias and enhanced enrichment of ribosome footprints relative to ribosomal RNA. Furthermore, P1 nuclease preserved a myriad of distinct juxtaposed ribosome complexes informative about yeast and human ribosome fates during translation initiation, stalling, and termination. Our optimized methods for mRNA footprint generation and capture provides a richer translatome profile using lower input and fewer technical challenges.

molecular biology↗

bioRxiv2022

Whole-genome resequencing reveals the population structure, genomic diversity, and demographic history of American chestnut (Castanea dentata)

American chestnut (Castanea dentata) was once the most economically and ecologically important hardwood species in the United States. In the first half of the 20th century, an exotic fungal pathogen - Cryphonectria parasitica - decimated the species, killing approximately four billion trees. Two approaches to developing blight resistant American chestnut populations show promise, but both will require introduction of adaptive genomic diversity from wild germplasm to produce diverse, locally adapted reforestation populations. Here we characterize population structure, demographic history, and genomic diversity in a range-wide sample of 384 wild American chestnuts to inform conservation and breeding with blight resistant varieties. Population structure analyses with DAPC and ADMIXTURE suggest that the chestnut range can be roughly divided into northeast, central, and southwest populations. Within-population genomic diversity estimates revealed a clinal pattern with the highest diversity in the southwest, which likely reflects bottleneck events associated with Quaternary glaciation. Finally, we identified genomic regions under positive selection within each population, which suggests that defense against fungal pathogens is a common target of selection across all populations. Taken together, these results show that American chestnut underwent a postglacial expansion from the southern portion of its range leading to three extant populations. These populations will serve as management units for breeding adaptive genetic variation into the blight-resistant tree populations for targeted reintroduction efforts.

genomics↗

bioRxiv2022

Deep sequencing of yeast and mouse tRNAs and tRNA fragments using OTTR

Among the major classes of RNAs in the cell, tRNAs remain the most difficult to characterize via deep sequencing approaches, as tRNA secondary structure and nucleotide modifications can both interfere with cDNA synthesis by commonly-used reverse transcriptases (RTs). Here, we benchmark a recently-developed RNA cloning protocol, termed Ordered Two-Template Relay (OTTR), to characterize intact tRNAs and tRNA fragments in budding yeast and in mouse tissues. We find that OTTR robustly captures full-length tRNAs in budding yeast and in mouse testis, with relatively low levels of premature termination at known barriers - 1-methylguanine, N2,N2-dimethylguanine, and 1-methyladenine - to typical reverse transcriptases. Moreover, these and several other nucleotide modifications leave misincorporation signatures in OTTR datasets which enables their detection without any additional protocol steps. Turning to analysis of small RNAs such as tRNA cleavage products, we compare OTTR with several standard small RNA-Seq protocols, finding that OTTR provides the most accurate picture of tRNA fragment levels by comparison to "ground truth" Northern blots. Applying this protocol to mature mouse spermatozoa, our data dramatically alter our understanding of the small RNA cargo of mature mammalian sperm, revealing a far more complex population of tRFs - including both 5 and 3 tRNA halves derived from the majority of tRNAs - than previously appreciated. Taken together, our data confirm the superior performance of OTTR to commercial protocols in analysis of tRNA fragments, and force a reappraisal of potential epigenetic functions of the sperm small RNA payload.

genomics↗

bioRxiv2021

Single-cell multi-omic velocity infers dynamic and decoupled gene regulation

Single-cell multi-omic datasets, in which multiple molecular modalities are profiled within the same cell, provide a unique opportunity to discover the relationships between cellular epigenomic and transcriptomic changes. To realize this potential, we developed MultiVelo, a mechanistic model of gene expression that extends the RNA velocity framework to incorporate epigenomic data. MultiVelo uses a probabilistic latent variable model to estimate the switch time and rate parameters of chromatin accessibility and gene expression from single-cell data, providing a quantitative summary of the temporal relationship between epigenomic and transcriptomic changes. Incorporating chromatin accessibility data significantly improves the accuracy of cell fate prediction compared to velocity estimates from RNA only. Fitting MultiVelo on single-cell multi-omic datasets from brain, skin, and blood cells reveals two distinct classes of genes distinguished by whether chromatin closes before or after transcription ceases. Our model also identifies four types of cell states-two states in which epigenome and transcriptome are coupled and two distinct decoupled states. The parameters inferred by MultiVelo quantify the length of time for which genes occupy each of the four states, ranking genes by the degree of coupling between transcriptome and epigenome. Finally, we identify time lags between transcription factor expression and binding site accessibility and between disease-associated SNP accessibility and expression of the linked genes. We provide an open-source Python implementation of MultiVelo on PyPI and GitHub (https://github.com/welch-lab/MultiVelo).

bioinformatics↗

bioRxiv2021

Low-bias ncRNA Libraries using Ordered Two-Template Relay: Serial Template Jumping by a Modified Retroelement Reverse Transcriptase

Non-long terminal repeat (non-LTR) and group II intron retroelements encode reverse transcriptases (RTs) that copy the retroelement transcript directly into host cell DNA, often at specific target sites. Biochemical characterization of these enzymes has been limited by recombinant expression and purification challenges, hampering understanding of their transposition mechanism and their exploitation for research and biotechnology. Properties of retroelement RTs substantiate their application for end-to-end RNA sequence capture. To investigate this utility, we first compared a non-LTR RT from Bombyx mori and a group II intron RT from Eubacterium rectale. Only the non-LTR RT showed processive template jumping, producing one cDNA from discontinuous templates each copied end-to-end. We also discovered an unexpected terminal deoxynucleotidyl transferase activity of the RTs that adds nucleotide(s) of choice to 3 ends of single-stranded RNA or DNA. Combining these two types of activity with additional insights about non-templated nucleotide additions to duplexed cDNA product, we developed a streamlined protocol for linking Next Generation Sequencing (NGS) adaptors to both cDNA ends in a single RT reaction. When benchmarked using a reference pool of microRNAs (miRNAs), library production using modified non-LTR retroelment RT for Ordered Two-Template Relay (OTTR) outperformed all commercially available kits and rivaled the low bias of technically demanding home-brew protocols. We applied OTTR to inventory RNAs purified from extracellular vesicles (EVs), identifying miRNAs as well as myriad other non-coding (nc) RNAs and ncRNA fragments. Our results establish the utility of OTTR for automation-friendly, low-bias, end-to-end RNA sequence inventories of complex ncRNA samples. SignificanceRetrotransposons are non-infectious mobile genetic elements that proliferate in host genomes via an RNA intermediate that is copied into DNA by a reverse transcriptase (RT) enzyme. RTs are important for biotechnological applications involving information capture from RNA, since RNA is first converted into complementary DNA for detection or sequencing. Here, we biochemically characterize RTs from two retroelements and uncover several activities that allowed us to design a streamlined, efficient workflow for determining the inventory of RNA sequences in processed RNA pools. The unique properties of non-retroviral RT activities obviate many technical issues associated with current methods of RNA sequence analysis, with wide applications in research, biotechnology, and diagnostics.

biochemistry↗

bioRxiv2020

Ultrastructural view of astrocyte-astrocyte and astrocyte-synapse contacts within the hippocampus

Astrocytes branch out and make contact at their interfaces. However, the ultrastructural interactions of astrocytes and astrocytes with their surroundings, including the spatial-location selectivity of astrocyte-synapse contacts, remain unknown. Here, the branching architecture of three neighboring astrocytes, their contact interfaces, and their surrounding neurites and synapses have been traced and 3D reconstructed using serial block-face scanning electron microscopy (SBF-SEM). Our reconstructions reveal extensive reflexive, loop-like processes that serve as scaffolds to neurites and give rise to spongiform astrocytic morphology. At the astrocyte-astrocyte interface, a cluster of process-process contacts were identified, which biophysically explains the existence of low inter-astrocytic electrical resistance. Additionally, we found that synapses uniformly made contact with the entire astrocyte, from soma to terminal processes, and can be ensheathed by two neighboring astrocytes. Lastly, in contrast to densely packed vesicles at the synaptic boutons, vesicle-like structures were scant within astrocytes. Together, these ultrastructural details should expand our understanding of functional astrocyte-astrocyte and astrocyte-neuron interactions.

neuroscience↗

bioRxiv2020

The orchestrated cellular and molecular responses of the kidney to endotoxin define the sepsis timeline

Clinical sepsis is a highly dynamic state that progresses at variable rates and has life-threatening consequences. Staging patients along the sepsis timeline requires a thorough knowledge of the evolution of cellular and molecular events at the tissue level. Here, we investigated the kidney, an organ central to the pathophysiology of sepsis. Single cell RNA sequencing revealed the involvement of various cell populations in injury and repair to be temporally organized and highly orchestrated. We identified key changes in gene expression that altered cellular functions and can explain features of clinical sepsis. These changes converged towards a remarkable global cell-cell communication failure and organ shutdown at a well-defined point in the sepsis timeline. Importantly, this time point was also a transition towards the emergence of recovery pathways. This rigorous spatial and temporal definition of murine sepsis will uncover precise biomarkers and targets that can help stage and treat human sepsis.

bioinformatics↗

bioRxiv2019

Reevaluation of the RNA binding properties of the Tetrahymena thermophila telomerase reverse transcriptase N-terminal domain

Telomerase restores chromosome-capping telomeric repeats lost with each round of genome replication by DNA-templated DNA polymerases. The telomerase reverse transcriptase (TERT) N-terminal (TEN) domain is a peripheral, telomerase-specific, processivity-stimulatory addition to more conserved domains that encircle the active site cavity. Reports of ciliate, yeast, and mammalian telomerase TEN domain associations with the telomerase RNA subunit (TR) describe low affinity interactions of uncertain specificity. Unfortunately two cryo-EM structures of synthesis-paused telomerase holoenzymes lack sufficient resolution to discriminate molecular specificity of possible TR contact(s) with the TEN domain, and there is no assigned density for the TEN domain termini implicated in RNA binding. Furthermore, studies have revealed alternative secondary structures for TR regions that could interact with TERT prior to TR folding into active conformation. Informed by recent advances in knowledge of telomerase structure, we returned to the investigation of Tetrahymena thermophila TERT TEN domain interaction with TR. Instead of finding specificity for a particular TR sequence or structure, we discovered that the tagged TEN domain used in previous characterizations has trace contamination with a bacterial RNA-interacting protein not detectable by SDS-PAGE. By resolving this interference, we show that the TEN domain binds RNAs dependent on RNA length rather than sequence.

biochemistry↗

bioRxiv2019

Gene Therapy for Follistatin Mitigates Systemic Metabolic Inflammation and Post-Traumatic Osteoarthritis in High-Fat Diet-Induced Obese Mice

Obesity-associated inflammation and loss of muscle function play critical roles in the development of osteoarthritis (OA); thus, therapies that target muscle tissue may provide novel approaches to restoring metabolic and biomechanical dysfunction associated with obesity. Recent studies indicate that follistatin (FST), a protein which binds myostatin and activin, may have the potential to enhance muscle formation while neutralizing inflammation induced by these proteins. Here, we hypothesized that adeno-associated virus (AAV9) delivery of FST will enhance muscle formation and mitigate metabolic inflammation and knee OA caused by a high fat diet in mice. Obese mice receiving AAV-mediated FST delivery exhibited decreased inflammatory adipokines and cytokines systemically in the serum as well as locally in the joint synovial fluid. Regardless of diet, mice receiving FST gene therapy were protected from post-traumatic OA and bone remodeling induced by joint injury. While obesity disrupted the mitochondrial oxidative phosphorylation (OXPHOS) system in adipocytes, gene therapy for FST restored the key proteins involved in mitochondrial biogenesis, such as PPAR{gamma} coactivator 1 and AKT protein kinase 1, leading to the browning of white adipose tissue. Taken together, these findings suggest that FST gene therapy may provide a multifactorial therapeutic approach for injury-induced OA and metabolic inflammation in obesity.

physiology↗

bioRxiv2019

A Genome-engineered Bioartificial Implant for Autoregulated Anti-Cytokine Drug Delivery

Biologic drug therapies are effective treatments for autoimmune diseases such as rheumatoid arthritis (RA) but may cause significant adverse effects, as they are administered continuously at high doses that can suppress the immune system. Using CRISPR-Cas9 genome editing, we engineered stem cells containing a synthetic gene circuit expressing biologic drugs to antagonize interleukin-1 (IL-1) or tumor necrosis factor (TNF) in an autoregulated, feedback-controlled manner in response to activation of the endogenous chemokine (C-C) motif ligand 2 (Ccl2) promoter. To test this approach in vivo, cells were tissue-engineered into a stable cartilaginous construct and implanted subcutaneously in mice with inflammatory arthritis. Bioengineered anti-cytokine implants mitigated arthritis severity as measured by joint pain, structural damage, and systemic and local inflammation. The coupling of synthetic biology with tissue engineering promises a range of potential applications for treating chronic diseases using custom-designed cells that express therapeutic transgenes in response to dynamically changing biological signals.Competing Interest StatementThe authors have declared no competing interest.View Full Text

synthetic biology↗

bioRxiv2018

Serotonin and neuropeptides are both released by the HSN command neuron to initiate C. elegans egg laying

1Neurons typically release both a small molecule neurotransmitter and one or more neuropeptides, but how these two types of signal from the same neuron might act together remains largely obscure. For example, serotonergic neurons in mammalian brain express the neuropeptide Substance P, but it is unclear how serotonin signaling might be modulated by a coreleased neuropeptide. We studied this issue in C. elegans, in which all serotonergic neurons express the neuropeptide NLP-3. The serotonergic Hermaphrodite Specific Neurons (HSNs) are command motor neurons within the egg-laying circuit that have previously been shown to release serotonin to initiate egg-laying behavior. We found that egg-laying defects in animals lacking serotonin were far milder than in animals lacking HSNs, suggesting that HSNs must release other signal(s) in addition to serotonin to stimulate egg laying. While null mutants for nlp-3 had only mild egg-laying defects, animals lacking both serotonin and NLP-3 had severe defects, like those of animals lacking HSNs. Optogenetic activation of HSNs induced egg laying in wild-type animals, or in mutant animals lacking either serotonin or NLP-3, but failed to induce egg laying in animals lacking both. We recorded calcium activity in the egg-laying muscles of animals lacking either serotonin, NLP-3, or both. The single mutants, and to a greater extent the double mutant, showed muscle activity that was uncoordinated and unable to expel eggs, such that the vm2 muscles cells that are direct postsynaptic targets of the HSN failed to contract simultaneously with other egg-laying muscle cells. Our results show that the HSN neurons use serotonin and the neuropeptide NLP-3 as partially redundant cotransmitters that together stimulate and coordinate activity of the target cells onto which they are released.

neuroscience↗

bioRxiv2018

Telomere length set point regulation in human pluripotent stem cells critically depends on the shelterin protein TPP1

Telomere maintenance is essential for the long-term proliferation of human pluripotent stem cells, while their telomere length set point determines the proliferative capacity of their differentiated progeny. The shelterin protein TPP1 is required for telomere stability and elongation, but its role in set point establishment remains elusive. Here, we characterize the contribution of TPP1 isoforms and residues outside the TEL patch, TPP1s telomerase interaction domain, to telomere length control. We demonstrate that TPP1L, the longer minor isoform of TPP1, can partially compensate for loss of the more abundant shorter isoform, TPP1S. Both TPP1S knockout and complete TPP1 knockout cell lines (TPP1 KO) show telomere shortening. However, TPP1S KO cells are able to stabilize short telomeres while TPP1 KO cells do not and die. We compare these phenotypes with that of TPP1L104A/L104A mutant cells that like the TPP1S KO have short stable telomeres. In contrast to TPP1S KO, TPP1L104A/L104A cells respond to increased telomerase. However, TPP1L104A/L104As sensitivity to shelterin-mediated feedback is altered, revealing TPP1L104A/L104A as a new type of shelterin mutant with aberrant set point regulation.

genetics↗